Methods of manufacturing glucose measuring assemblies with hydrogels

ABSTRACT

This invention relates to methods for reducing the presence of a compound in an ionically conductive material, e.g., for use in iontophoretic devices, wherein the presence of the compound interferes with detecting a selected analyte. Removal of the compound can typically take place either during or after the manufacture of the ionically conductive material or an assembly comprising this material. Also disclosed are methods for generating selectively permeable barriers on the reactive faces of electrodes. Further, this invention relates to hydrogels comprising one or more biocides, as well as assemblies containing such hydrogels.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.10/438,239, filed 14 May 2003, now U.S. Pat. No. 6,902,905, which is acontinuation of U.S. patent application Ser. No. 09/556,486 filed 21Apr. 2000, now U.S. Pat. No. 6,615,078, which claims the benefit of U.S.Provisional Patent Application Ser. Nos. 60/130,729, filed 22 Apr. 1999,and 60/149,513, filed 17 Aug. 1999, all of which applications are hereinincorporated by reference in their entireties.

TECHNICAL FIELD

This invention relates generally to methods and devices for reducing thepresence of a biocide in an ionically conductive material, e.g., for usein iontophoretic devices, either during or after the manufacture of theionically conductive material or an assembly comprising this material.In addition, this invention relates to hydrogels comprising one or morebiocides.

BACKGROUND

A number of diagnostic tests are routinely performed on humans toevaluate the amount or existence of analytes present in blood or otherbody fluids. These diagnostic tests typically rely on physiologicalfluid samples removed from a subject, either using a syringe or bypricking the skin.

PCT Publication No. WO 96/00110, published 4 Jan. 1996, describes aniontophoretic apparatus for transdermal monitoring of a target analyte,wherein an iontophoretic electrode is used to move the analyte into acollection reservoir and a biosensor is used to detect the analyte. InU.S. Pat. No. 5,279,543 to Glikfeld, iontophoresis is used to sample asubstance through skin and into a receptacle on the skin surface.Glikfeld suggests that this sampling procedure can be coupled with aglucose-specific biosensor or glucose-specific electrodes in order tomonitor blood glucose. Additionally, U.S. Pat. Nos. 5,362,307 and5,730,714 both to Guy, et al. describe sampling devices.

Analytical biosensors have been embraced during the last decade as ameans of combining the advantages of electrochemical signal transductionwith the specificity inherent in biological interactions. However, twofactors that may affect the quality of the data generated by the signaltransduction are as follows. First, compounds unrelated to the analyteof interest may enter the analytical system and interact directly withthe electrode assembly, leading to signal generation unrelated to theconcentration of the analyte or its derivatives. These interferingspecies may be introduced either during manufacture of the biosensor orduring its use. For example, certain compounds present in sample fluid(e.g., acetominophen and uric acid) are electrochemically “active” andare capable of signal generation independent of the specific biologicalsystem employed by the biosensor, via a direct interaction with theelectrode. Additionally, compounds that may interact at an electrode mayhave been introduced during manufacturing for specific purposes, such asto provide antimicrobial or antifungal activity (biocides). Theseinterfering species may produce overlapping current signals, thusdecreasing the selectivity of the biosensor. Additionally, the compoundsmay irreversibly bind to the reactive face of the electrode assembly,leading to fouling of the sensing surface and reduced sensitivity.

Several techniques have been employed to minimize the effects ofinterfering species on electrode function to get around these issues.One technique is to use the lowest polarizing voltage sufficient for theintended reaction. This reduces the current (i.e., electrons) generatedby any undesired electrochemical oxidations requiring polarizingvoltages higher than what is required for the intended reaction.However, because some enzymatic systems employed in biosensors requirevoltage levels that do not provide sufficient screening of signalsgenerated by interfering species, the voltage level cannot be decreasedbelow that which allows generation of signals from the interferingspecies.

A second technique has been to construct membranes or other physicalbarriers to impede the interfering species from reaching the face of theelectrode. The list of films which may be employed includes celluloseacetate, poly(o-phenylenediamine), polyphenol, polypyrrole,polycarbonate, and Nafion® (E.I du Pont de Nemours & Co., WilmingtonDel.) polymer. However, such membranes can be difficult to prepare andmay not efficiently attach to the reactive surface of the electrode.There remains a need in the art for methods and devices which provide anefficient reduction of interfering species while maintaining efficientdetection of an analyte.

SUMMARY OF THE INVENTION

The present invention provides methods and devices for reducing thepresence of a compound in an ionically conductive material wherein thepresence of the compound interferes with detecting an analyte in thematerial. By decreasing the level of interfering species present in theionically conductive material, the present invention increases thepercentage of signal that arises from an analyte of interest (or itsderivatives) during use of a sampling device. In one aspect of thepresent invention, the reduction in interferant signal is achieved byselectively adsorbing the interfering compound from the ionicallyconductive material before the compound can reach the sensor means andgenerate a signal. In a second aspect of the invention, the interferingspecies are reduced by polymerizing an interfering compound to form anelectrochemically-inactive but permeation selective barrier at thereactive face of the sensor means. The permeation selectivecharacteristics of the polymer barrier can provide the added benefit ofreducing signals generated from interferants other than the speciesbeing polymerized. Because the aforementioned permeation selectivebarrier is created on the reactive face of the sensor means in ratherthan prior to construction of the collection assembly, the presentinvention provides efficient means for manufacturing collectionassemblies that use this method for reducing the presence of aninterferant compound.

Accordingly, it is a general object of the invention to provide a methodfor reducing the presence of a compound in an ionically conductivematerial wherein the presence of the compound interferes with detectingan analyte in the material. In one embodiment, the method includesplacing the material containing the compound in contact with at leastone component of a device used for detecting the analyte, wherein thecomponent is partially permeable to the compound. The component and thecompound are contacted under conditions that allow the compound tomigrate out of the material and into the component, thus reducing thepresence of the compound in the material. In the present invention, thecomponent is preferably composed of a polyurethane-like material or apolyester-like material.

In another embodiment of the present invention, the presence of aninterfering compound is reduced essentially as follows. The ionicallyconductive material containing the interfering compound is placed incontact with a reactive face of a sensor element (for example, a sensorelectrode). The ionically conductive material and the sensing elementare arranged such that when a current is flowing to the sensing element,the current flows through the ionically conductive material containingthe compound. The sensor element is then activated to provide anelectrical current for a period of time and under conditions sufficientto polymerize the compound on the reactive face of the sensor. Previousapproaches for forming permeably selective films on electrodes requiredthat the film was formed ex situ, that is before use, and the presentinvention demonstrates that the permeably selective barrier can beformed in situ. In the present invention, a preferred group ofpolymerizable interferant compounds are phenolic compounds, for examplethe p-hydroxybenzoic acid esters commonly referred to as “parabens.”

In a further embodiment of the invention, a method of forming apermeation-selective barrier on an electrode face in situ is described,the method comprising the steps of a) formulating an ionicallyconductive material comprising a phenolic compound capable ofpolymerizing under the influence of an electrical current, b) placingthe material in contact with a reactive face of a sensing electrode suchthat when current is flowing to the electrode current flows through thematerial, and c) activating the electrode to provide an electricalcurrent for a period of time and under conditions sufficient topolymerize the compound on the reactive face of the sensor and form apermeation-selective barrier. In the present invention, a preferredgroup of phenolic compounds are the p-hydroxybenzoic acid esterscommonly referred to as “parabens.”

In another embodiment of the present invention, a collection assemblyfor use in a sampling system is described. The collection assembly iscomprised of a collection insert layer containing an ionicallyconductive material, wherein the ionically conductive material containsa compound that will polymerize on the reactive face of a sensor elementplaced adjacent to the ionically conductive material. Also described isa method of manufacturing a collection assembly The method ofmanufacture of the collection assembly comprises the steps of a) formingthe ionically conductive medium containing the interfering compound, b)contacting one surface of the ionically conductive medium with a masklayer composed of a material that is substantially impermeable to theselected analyte or derivatives thereof, and c) contacting a secondsurface of the ionically conductive medium with a retaining layer toform the collection assembly.

In a further embodiment of the present invention, an autosensor assemblyfor use in a sampling system is described. The autosensor assembly iscomprised of a) a collection insert layer containing an ionicallyconductive medium, an enzyme capable of reacting with an analyte toproduce hydrogen peroxide, and a phenolic compound which will polymerizeunder an electric current; and b) a sensor element in operative contactwith the collection insert layer, positioned such that the phenoliccompound can react electrochemically with the reactive face of thesensor element to provide a selectively permeable barrier at aninterface between the sensor element and the collection insert layer.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded pictorial representation of components from anexemplary sampling system.

FIG. 2 depicts the response of sensor electrodes to various analytes inhydrogels in the presence or absence of phenolic compounds. Charge(y-axis) is depicted at various time intervals (x-axis). The solidsquares with solid connecting lines depict the sensor electrodes'response to acetaminophen for a system containing a standard gel (i.e.,without biocide); solid squares with dashed connecting lines representsthe electrodes' response to acetaminophen in the presence of a phenolicgel; solid triangles with solid connecting lines represent theelectrodes' response to glucose for a standard gel; solid triangles withdashed connecting lines represent the electrodes' response to glucose inthe presence of a phenolic gel; solid double-triangles with solidconnecting lines represent the electrodes' response to H₂O₂ in astandard gel; solid double-triangles with dashed connecting linesrepresent the electrodes' response to H₂O₂ in a phenolic gel; solidcircles with solid connecting lines represent the electrodes' responseto uric acid in a standard gel; and solid circles with dashed connectinglines represent the electrodes' response to uric acid in a phenolic gel.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular compositionsor biological systems as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a”, “an” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “areservoir” includes a combination of two or more such reservoirs,reference to “an analyte” includes mixtures of analytes, and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

The terms “analyte” and “target analyte” are used herein to denote anyphysiological analyte of interest that is a specific substance orcomponent that is being detected and/or measured in a chemical,physical, enzymatic, or optical analysis. A detectable signal (e.g., achemical signal or electrochemical signal) can be obtained, eitherdirectly or indirectly, from such an analyte or derivatives thereof.Furthermore, the terms “analyte” and “substance” are usedinterchangeably herein, and are intended to have the same meaning, andthus encompass any substance of interest. In preferred embodiments, theanalyte is a physiological analyte of interest, for example, glucose, ora chemical that has a physiological action, for example, a drug orpharmacological agent.

The term “interferant” or “interfering species” refers to anelectroactive compound other than the analyte of interest which, whenpresent in an ionically conductive material, generates a responseunrelated to the concentration (or amount) of analyte being measured bythe sampling system, thus interfering with the detection of an analytein the material.

The term “biocide” is used herein to describe any substance that killsor inhibits the growth of micro-organisms, including but not limited to,viruses, bacteria, molds, slimes, yeast and fungi. A biocide may be amaterial that is also toxic to humans, but is preferably a materialwhich, when used in relatively low concentrations, in an ionicallyconductive material such as a patch or a hydrogel, does not cause skinirritation or any adverse effects on the human subject.

A “sampling device,” “sampling mechanism” or “sampling system” refers toany device for obtaining a sample from a biological system for thepurpose of determining the concentration of an analyte of interest. Such“biological systems” include any biological system from which theanalyte of interest can be extracted, including, but not limited to,blood, interstitial fluid, perspiration and tears. Further, a“biological system” includes both living and artificially maintainedsystems. As used herein, the term “sampling” mechanism refers toextraction of a substance from the biological system, generally across amembrane such as the stratum corneum or mucosal membranes, by invasive,minimally invasive, or non-invasive means. The membrane can be naturalor artificial, and can be of plant or animal nature, such as natural orartificial skin, blood vessel tissue, intestinal tissue, and the like.Typically, the sampling mechanism are in operative contact with a“reservoir,” or “collection reservoir,” wherein the sampling mechanismis used for extracting the analyte from the biological system into thereservoir to obtain the analyte in the reservoir. Non-limiting examplesof sampling techniques include iontophoresis, sonophoresis (see, e.g.,International Publication No. WO 91/12772, published 5 Sep. 1991),suction, electroporation, thermal poration, passive diffusion (see,e.g., International Publication Nos.: WO 97/38126 (published 16 Oct.1997); WO 97/42888, WO 97/42886, WO 97/42885, and WO 97/42882 (allpublished 20 Nov. 1997); and WO 97/43962 (published 27 Nov. 1997),microfine (miniature) lances or cannulas, subcutaneous implants orinsertions, and laser devices (see, e.g., Jacques et al. (1978) J.Invest. Dermatology 88: 88–93; International Publication WO 99/44507,published 1999 Sep. 10; International Publication WO 99/44638, published1999 Sep. 10; and International Publication WO 99/40848, published 1999Aug. 19). Iontophoretic sampling devices are described, for example, inInternational Publication No. WO 97/24059, published 10 Jul. 1997;European Patent Application EP 0942 278, published 15 Sep. 1999;International Publication No. WO 96/00110, published 4 Jan. 1996;International Publication No. WO 97/10499, published 2 Mar. 1997; U.S.Pat. Nos. 5,279,543; 5,362,307; 5,730,714; 5,771,890; 5,989,409;5,735,273; 5,827,183; 5,954,685 and 6,023,629, all of which are hereinincorporated by reference in their entireties.

The term “physiological fluid” as used herein refers to any desiredfluid to be sampled, and includes, but is not limited to, blood,cerebrospinal fluid, interstitial fluid, semen, sweat, saliva, urine andthe like.

The term “artificial,” as used herein, refers to an aggregation of cellsof monolayer thickness or greater which are grown or cultured in vivo orin vitro, and which function as a tissue of an organism but are notactually derived, or excised, from a pre-existing source or host.

A “monitoring system,” as used herein, refers to a system useful forfrequently measuring a physiological analyte present in a biologicalsystem. Such a system typically includes, but is not limited to,sampling mechanism, sensing mechanism, and a microprocessor mechanism inoperative communication with the sampling mechanism and the sensingmechanism.

As used herein, the term “frequent measurement” intends a series of twoor more measurements obtained from a particular biological system, whichmeasurements are obtained using a single device maintained in operativecontact with the biological system over a time period in which a seriesof measurements (e.g, second, minute or hour intervals) is obtained. Theterm thus includes continual and continuous measurements.

The term “subject” encompasses any warm-blooded animal, particularlyincluding a member of the class Mammalia such as, without limitation,humans and nonhuman primates such as chimpanzees and other apes andmonkey species; farm animals such as cattle, sheep, pigs, goats andhorses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs, and the like. Theterm does not denote a particular age or sex and, thus, includes adultand newborn subjects, whether male or female.

The term “transdermal,” as used herein, includes both transdermal andtransmucosal techniques, i.e., extraction of a target analyte acrossskin, e.g., stratum corneum, or mucosal tissue. Aspects of the inventionwhich are described herein in the context of “transdermal,” unlessotherwise specified, are meant to apply to both transdermal andtransmucosal techniques.

The term “transdermal extraction,” or “transdermally extracted” intendsany sampling method, which entails extracting and/or transporting ananalyte from beneath a tissue surface across skin or mucosal tissue. Theterm thus includes extraction of an analyte using, for example,iontophoresis (reverse iontophoresis), electroosmosis, sonophoresis(see, e.g., U.S. Pat. No. 5,636,632), microdialysis, suction, andpassive diffusion. These methods can, of course, be coupled withapplication of skin penetration enhancers or skin permeability enhancingtechnique such as various substances or physical methods such as tapestripping or pricking with micro-needles. The term “transdermallyextracted” also encompasses extraction techniques which employ thermalporation, laser microporation, electroporation, microfine lances,microfine canulas, subcutaneous implants or insertions, and the like.

The term “iontophoresis” intends a method for transporting substancesacross tissue by way of an application of electrical energy to thetissue. In conventional iontophoresis, a reservoir is provided at thetissue surface to serve as a container of (or containment means for)material to be transported. Iontophoresis can be carried out usingstandard methods known to those of skill in the art, for example byestablishing an electrical potential using a direct current (DC) betweenfixed anode and cathode “iontophoretic electrodes,” alternating a directcurrent between anode and cathode iontophoretic electrodes, or using amore complex waveform such as applying a current with alternatingpolarity (AP) between iontophoretic electrodes (so that each electrodeis alternately an anode or a cathode). For example, see U.S. Pat. Nos.5,771,890 and 6,023,629 and PCT Publication No. WO 96/00109, published 4Jan. 1996.

The term “reverse iontophoresis” refers to the movement of a substancefrom a biological fluid across a membrane by way of an applied electricpotential or current. In reverse iontophoresis, a reservoir is providedat the tissue surface to receive the extracted material, as used in theGlucoWatch® (Cygnus, Inc., Redwood City, Calif.) glucose monitor (See,e.g., Tamada et al. (1999) JAMA 282: 1839–1844).

“Electroosmosis” refers to the movement of a substance through amembrane by way of an electric field-induced convective flow. The termsiontophoresis, reverse iontophoresis, and electroosmosis, will be usedinterchangeably herein to refer to movement of any ionically charged oruncharged substance across a membrane (e.g., an epithelial membrane)upon application of an electric potential to the membrane through anionically conductive medium.

The term “sensing device,” “sensing mechanism,” or “biosensor device”encompasses any device that can be used to measure the concentration oramount of an analyte, or derivative thereof, of interest. Preferredsensing devices for detecting blood analytes generally includeelectrochemical devices, optical and chemical devices and combinationsthereof. Examples of electrochemical devices include the Clark electrodesystem (see, e.g., Updike, et al., (1967) Nature 214: 986–988), andother amperometric, coulometric, or potentiometric electrochemicaldevices. Examples of optical devices include conventional enzyme-basedreactions as used in the Lifescan® (Johnson and Johnson, New Brunswick,N.J.) glucose monitor (see, e.g., U.S. Pat. No. 4,935,346 to Phillips,et al.).

A “biosensor” or “biosensor device” includes, but is not limited to, a“sensor element” which includes, but is not limited to, a “biosensorelectrode” or “sensing electrode” or “working electrode” which refers tothe electrode that is monitored to determine the amount of electricalsignal at a point in time or over a given time period, which signal isthen correlated with the concentration of a chemical compound. Thesensing electrode comprises a reactive surface which converts theanalyte, or a derivative thereof, to electrical signal. The reactivesurface can be comprised of any electrically conductive material suchas, but not limited to, platinum-group metals (including, platinum,palladium, rhodium, ruthenium, osmium, and iridium), nickel, copper,silver, and carbon, as well as, oxides, dioxides, combinations or alloysthereof. Some catalytic materials, membranes, and fabricationtechnologies suitable for the construction of amperometric biosensorsare described by Newman, J. D., et al. (1995) Analytical Chemistry 67:4594–4599.

The “sensor element” can include components in addition to the sensingelectrode, for example, it can include a “reference electrode” and a“counter electrode.” The term “reference electrode” is used herein tomean an electrode that provides a reference potential, e.g., a potentialcan be established between a reference electrode and a workingelectrode. The term “counter electrode” is used herein to mean anelectrode in an electrochemical circuit that acts as a current source orsink to complete the electrochemical circuit. Although it is notessential that a counter electrode be employed where a referenceelectrode is included in the circuit and the electrode is capable ofperforming the function of a counter electrode, it is preferred to haveseparate counter and reference electrodes because the referencepotential provided by the reference electrode is most stable when it isat equilibrium. If the reference electrode is required to act further asa counter electrode, the current flowing through the reference electrodemay disturb this equilibrium. Consequently, separate electrodesfunctioning as counter and reference electrodes are preferred.

In one embodiment, the “counter electrode” of the “sensor element”comprises a “bimodal electrode.” The term “bimodal electrode” as usedherein typically refers to an electrode which is capable of functioningnon-simultaneously as, for example, both the counter electrode (of the“sensor element”) and the iontophoretic electrode (of the “samplingmechanism”) as described, for example, U.S. Pat. No. 5,954,685.

The terms “reactive surface,” and “reactive face” are usedinterchangeably herein to mean the surface of the sensing electrodethat: (1) is in contact with the surface of an ionically conductivematerial which contains an analyte or through which an analyte, or aderivative thereof, flows from a source thereof; (2) is comprised of acatalytic material (e.g., carbon, platinum, palladium, rhodium,ruthenium, or nickel and/or oxides, dioxides and combinations or alloysthereof) or a material that provides sites for electrochemical reaction;(3) converts a chemical signal (for example, hydrogen peroxide) into anelectrical signal (e.g., an electrical current); and (4) defines theelectrode surface area that, when composed of a reactive material, issufficient to drive the electrochemical reaction at a rate sufficient togenerate a detectable, reproducibly measurable, electrical signal thatis correlatable with the amount of analyte present in the electrolyte.

An “ionically conductive material” refers to any material that providesionic conductivity, and through which electrochemically active speciescan diffuse. The ionically conductive material can be, for example, asolid, liquid, or semi-solid (e.g., in the form of a gel) material thatcontains an electrolyte, which can be composed primarily of water andions (e.g., sodium chloride), and generally comprises 50% or more waterby weight. The material can be in the form of a hydrogel, a sponge orpad (e.g., soaked with an electrolytic solution), or any other materialthat can contain an electrolyte and allow passage of electrochemicallyactive species, especially the analyte of interest.

The term “buffer” refers to one or more components which are added to acomposition in order to adjust or maintain the pH of the composition.

The term “electrolyte” is used herein to a component of the ionicallyconductive medium which allows for an ionic current to flow within themedium. This component of the ionically conductive medium can be one ormore salts or buffer components, but is not limited to these materials.

The term “humectant” is used herein to describe a substance which has anaffinity for water or a stabilizing effect on the water content of acomposition.

The term “collection reservoir” is used to describe any suitablecontainment means for containing a sample extracted from a biologicalsystem. For example, the collection reservoir can be a receptaclecontaining a material which is ionically conductive (e.g., water withions therein), or alternatively it can be a material, such as asponge-like material or hydrophilic polymer, used to keep the water inplace. Such collection reservoirs can be in the form of a hydrogel (forexample, in the shape of a disk or pad). Hydrogels are typicallyreferred to as “collection inserts.” Other suitable collectionreservoirs include, but are not limited to, tubes, vials, strips,capillary collection devices, cannulas, and miniaturized etched, ablatedor molded flow paths.

A “collection insert layer” is a layer of an assembly or laminatecomprising a collection reservoir (or collection insert) located, forexample, between a mask layer and a retaining layer.

The term “permeation selective” or “permselective” refers to a propertyof a membrane barrier wherein passage through the membrane is selective,depending upon the physical and chemical properties of the membrane aswell as those of the compound involved. For example, permselective filmsallow the transport of an analyte or its derivatives, while preventingundesirable compounds (interferants) from passing. (See, for instance,Chapter 10, “Permselective Coatings for Amperometric Biosensing” in ACSSymposium Series No. 487 (1992) American Chemical Society.)

A “laminate”, as used herein, refers to structures comprised of at leasttwo bonded layers. The layers may be bonded by welding or through theuse of adhesives. Examples of welding include, but are not limited to,the following: ultrasonic welding, heat bonding, and inductively coupledlocalized heating followed by localized flow. Examples of commonadhesives include, but are not limited to, pressure sensitive adhesives,thermoset adhesives, cyanocrylate adhesives, epoxies, contact adhesives,and heat sensitive adhesives.

A “collection assembly”, as used herein, refers to structures comprisedof several layers, where the assembly includes at least one collectioninsert layer, for example a hydrogel. An example of a collectionassembly as referred to in the present invention is a mask layer,collection insert layer, and a retaining layer where the layers are heldin appropriate functional relationship to each other but are notnecessarily a laminate (i.e., the layers may not be bonded together. Thelayers may, for example, be held together by interlocking geometry orfriction).

The term “mask layer” as used herein refers to a component of acollection assembly that is substantially planar and typically contactsboth the biological system and the collection insert layer. See, forexample, U.S. Pat. Nos. 5,735,273, and 5,827,183, herein incorporated byreference.

The term “gel retaining layer” or “gel retainer” as used herein refersto a component of a collection assembly that is substantially planar andtypically contacts both the collection insert layer and the electrodeassembly.

The term “support tray” as used herein typically refers to a rigid,substantially planar platform and is used to support and/or align theelectrode assembly and the collection assembly. The support trayprovides a means for placing the electrode assembly and the collectionassembly into the sampling system.

An “autosensor assembly”, as used herein, refers to a structuregenerally comprising a mask layer, collection insert layer, a gelretaining layer, an electrode assembly, and a support tray. Theautosensor assembly may also include liners where the layers are held inapproximate, functional relationship to each other. Exemplary collectionassemblies and autosensor structures are described, for example, inInternational Publication WO 99/58190, published 18 Nov. 1999; and U.S.Pat. Nos. 5,735,273 and 5,827,183. The mask and retaining layers arepreferably composed of materials that are substantially impermeable tothe analyte (chemical signal) to be detected; however, the material canbe permeable to other substances. By “substantially impermeable” ismeant that the material reduces or eliminates chemical signal transport(e.g., by diffusion). The material can allow for a low level of chemicalsignal transport, with the proviso that chemical signal passing throughthe material does not cause significant edge effects at the sensingelectrode.

The term “in situ” refers to the location of an occurrence with respectto an original position. In the case of the present invention, the termrefers to the formation of a permselective polymer barrier on thereactive face of a sensing element, this being the original position orplace of contact between the sensing element and the ionicallyconductive material comprising the compound to be polymerized.

The terms “about” or “approximately” when associated with a numericvalue refers to that numeric value plus or minus 10 units of measure(i.e. percent, grams, degrees or volts), preferably plus or minus 5units of measure, more preferably plus or minus 2 units of measure, mostpreferably plus or minus 1 unit of measure.

By the term “printed” as used herein is meant a substantially uniformdeposition of an electrode formulation onto one surface of a substrate(i.e., the base support). It will be appreciated by those skilled in theart that a variety of techniques may be used to effect substantiallyuniform deposition of a material onto a substrate, e.g., Gravure-typeprinting, extrusion coating, screen coating, spraying, painting, or thelike.

The term “physiological effect” encompasses effects produced in thesubject that achieve the intended purpose of a therapy. In preferredembodiments, a physiological effect means that the symptoms of thesubject being treated are prevented or alleviated. For example, aphysiological effect would be one that results in the prolongation ofsurvival in a patient.

2. General Methods, Biocides, and Formulations

Methods and devices for reducing the presence of a compound in anionically conductive material wherein the presence of the compoundinterferes with detecting an analyte in the material are provided bythis invention. Further included in the present invention is anapparatus incorporating the methods and devices described herein. Themethods and apparatus may be employed in a sampling system, to enhancethe detection and/or quantification of the concentration of a targetanalyte present in a biological system. Although the methods andapparatus are broadly applicable to sampling any chemical analyte and/orsubstance, the preferred embodiment of the invention is used intransdermal sampling and quantifying or qualifying glucose or a glucosemetabolite.

As will be understood by the ordinarily skilled artisan upon reading thespecification, the analyte can be any specific substance or componentthat one is desirous of detecting and/or measuring in a chemical,physical, enzymatic, or optical analysis. Such analytes include, but arenot limited to, amino acids, enzyme substrates or products indicating adisease state or condition, other markers of disease states orconditions, drugs of abuse (e.g., ethanol, cocaine), therapeutic and/orpharmacologic agents, electrolytes, physiological analytes of interest(e.g., calcium, potassium, sodium, chloride, bicarbonate (CO₂), glucose,urea (blood urea nitrogen), lactate or lactic acid, hematocrit, andhemoglobin), lipids, and the like. In preferred embodiments, the analyteis a physiological analyte of interest, for example glucose, or achemical that has a physiological action, for example a drug orpharmacological agent.

During manufacture of the autosensor assembly, one or more biocides maybe incorporated into the ionically conductive material. Biocides ofinterest for the methods of the present invention include, but are notlimited to, compounds such as chlorinated hydrocarbons; organometallics;hydrogen releasing compounds; metallic salts; organic sulfur compounds;phenolic compounds (including but not limited to a variety of NipaHardwicke Inc. liquid preservatives registered under the trade namesNipastat®, Nipaguard®, Phenosept®, Phenonip®, Phenoxetol®, andNipacide®); quartenary ammonium compounds; surfactants and othermembrane-disrupting agents (including but not limited to undecylenicacid and its salts), and the like. However, the biocides often act asinterfering species. The present disclosure teaches formulationsincorporating biocides into components of an autosensor assembly as wellas methods of removing such biocides after manufacture of the autosensorassembly and assembly components.

One biocide used in the practice of the present invention is undecylenicacid (10-undecenoic acid, or UA). Undecylenic acid is an unsaturatedfatty acid which has been used since the 1940's as an relativelynonirritating and reasonably effective treatment for preventing thegrowth of pathogenic organisms on the skin. Both the acid form(“undecylenic acid”) and the salt forms (“undecylenates”) have biocidicactivity, and may be used in combination with one another (or with otherbiocides). The biocide is commonly referred to herein as “undecylenicacid” without differentiation between the acid and salt forms. The saltforms may include but are not limited to the sodium, calcium and zincsalts. In addition, other esters of undecylenate, including but notlimited to the methyl, ethyl, propyl, isopropyl, glyceryl, benzyl, allyland epoxypropyl esters, are effective as biocides. When used as abiocide in the hydrogels of the present invention, the undecylenatebiocide (acid, salt or mixture thereof) is present in the hydrogel at aconcentration high enough to be effective as a biocide, for examplebetween about 0.001 wt % and about 10 wt %, preferably between about0.01 wt % and about 5 wt %, more preferably between about 0.1 wt % andabout 2 wt %.

Another preferred biocide is Nipastat® sodium p-hydrozybenzoic acidesters (Nipa Hardwicke, Inc., Wilmington Del.). Nipastat® biocide is amixture of sodium derivatives of p-hydroxybenzoate. The major componentof the mixture is methyl paraben (methyl p-hydroxybenzoate) with minorcomponents of the ethyl-, propyl-, butyl-, andiso-butyl-p-hydroxybenzoates. Any such parabens can be used in thepractice of the present invention, individually or preferably inmixtures. In addition, mixtures of different types of biocides can beused (e.g., parabens plus other biocides). When used as a biocide in thehydrogels of the present invention, the Nipastat® biocide is present inthe hydrogel at a concentration high enough to be effective as abiocide, for example between about 0.001 wt % and about 10 wt %,preferably between about 0.01 wt % and about 5 wt %, more preferablybetween about 0.1 wt % and about 2 wt %.

Experiments performed in support of the present invention show thatthese biocides, when incorporated into a collection reservoir orcollection reservoir material (e.g., a hydrogel), are effective biocidesagainst a number of microbial organisms, including, but not limited to,Aspergillus niger, Candida albicans, Eschericia coli, Pseudomonasaeruginosa and Staphylococcus aureus.

The collection reservoir typically contains an ionically conductiveliquid or liquid-containing medium. In one embodiment, the collectionreservoir is preferably a hydrogel which can contain ionic substances,or electrolytes, in an amount sufficient to produce high ionicconductivity. The hydrogel is formed from a solid material (solute)which, when combined with water, forms a gel by the formation of astructure which holds water including interconnected cells and/ornetwork structure formed by the solute. Suitable hydrogel formulationsare described in PCT Publication Nos. WO 97/02811, published 30 Jan.1997, and WO 96/00110, published 4 Jan. 1996. The solute may be anaturally occurring material such as the solute of natural gelatin whichincludes a mixture of proteins obtained by the hydrolysis of collagen byboiling skin, ligaments, tendons and the like. However, the solute orgel forming material is more preferably a polymer material (including,but not limited to, polyethylene oxide, polyvinyl alcohol, polyacrylicacid, polyacrylamidomethylpropanesulfonate and mixtures and/orcopolymers thereof) present in an amount in the range of more than 0.5%and less than 40% by weight, preferably 8 to 12% by weight when ahumectant is also added, and preferably about 15 to 20% by weight whenno humectant is added.

While not required, crosslinking of the polymer may be performed toimprove the structural integrity of the hydrogel. The crosslinking maybe achieved by thermal reaction, chemical reaction or by providingionizing radiation (for example, electron beam radiation, UV radiationor gamma radiation). Various agents which can be used to facilitatecrosslinking within a polymer in conjunction with ionizing radiation aredisclosed in U.S. Pat. Nos. 4,684,558 and 4,989,607 incorporated hereinby reference. Crosslinkers which may be used in the present inventioninclude but are not limited to N,N-methylenebisacrylamide, polypropyleneglycol monomethacrylate, polypropylene glycol monoacrylate, polyethyleneglycol dimethacrylate, polyethylene glycol diacrylate,triallylisocyanurate (TAIC), diallylisocyanurate (DAIC), triacrylatessuch as SR 454 ethoxylated trimethylolpropane triacrylate, and SR 9035highly alkoxylated trimethylolpropane triacrylate, available fromSartomer (Exton, Pa.), ethylene glycol methacrylate, triethylene glycolmethacrylate, trimethylolpropane trimethacrylates, and glutaraldehyde.Furthermore, a photoinitiator may be used to facilitate the crosslinkingprocess.

In addition to crosslinking of the polymer, the ionically conductivemedium of the present invention may comprise a structural support whichis embedded in the hydrogel. This support includes, but is not limitedto, a woven fabric, a nonwoven fabric, dispersed fibers, or a membrane.The ionically conductive medium can be polymerized separately, or in thepresence of this “scrim” or nonwoven material such as polyester orpolypropylene. Two exemplary nonwoven materials are Delnet® nonwoven andRemay® nonwoven, available from AET Specialty Nets.

Additional materials may be added to the hydrogel, including, withoutlimitation, one or more electrolytes (e.g., salts), buffers, tackifiers,humectants, crosslinkers, biocides, preservatives, chelators (forexample, ethylenediamine tetraacetic acid) and enzyme stabilizers. Avariety of buffers may be used in connection with the present invention,including but not limited to various salts of phosphate, citrates,bicarbonates, succinates, acetates and lactates. One preferred buffer isphosphate buffer. The buffer is preferably present in amounts tomaintain the pH of the hydrogel in a range of about pH 3–9, morepreferably pH 6–8. A preferred electrolyte is sodium chloride, but othersalts may be equally employed. Humectants useful in the presentinvention include, but are not limited to, glycerol, hexylene glycol andsorbitol.

In one aspect, the present invention relates to hydrogels containing abiocide of interest. For example, a hydrogel, comprises,

(a) a hydrophilic compound which forms a gel in the presence of water,which compound is present in an amount of about 4% or more by weightbased on the total weight of the hydrogel;

(b) water in an amount of about 95% or less based on the total weight ofthe hydrogel;

(c) an electrolyte, wherein background electrical signal in the gel isless than approximately 200 nA;

(d) an enzyme composition; and

(e) a biocide.

Exemplary biocides include, but are not limited to chlorinatedhydrocarbons, organometallics, hydrogen releasing compounds, metallicsalts, quaternary ammonium compounds, organic sulfur compounds,phenolics, and methylparabens. Preferred biocides of the presentinvention include undecylenates (e.g., undecylenic acid, a salt ofundecylenic acid, or mixtures thereof), and parabens. Biocides may be,for example, antimicrobial and/or antifungal.

Typically, the background electrical signal in a gel is in the range ofabout 20 to about 250 nA, preferably between about 25 to about 100 nA,more preferably between about 30 and about 90 nA, for example, about 50nA.

Exemplary enzyme compositions are discussed herein. Use of a selectedenzyme depends on the analyte which is to be detected. In oneembodiment, for the detection of glucose, such an enzyme is glucoseoxidase. The glucose oxidase may be present in an amount of from about10 units to about 5,000 units per gram of the total weight of thehydrogel, preferably approximately 200 units or more. Degradativecomponents of the enzyme composition are reduced such that quantitationof the analyte is not compromised, for example, the glucose oxidase cancatalyze a reaction between glucose and oxygen resulting in thegeneration of hydrogen peroxide; accordingly, the hydrogen peroxidedegradative components of the enzyme composition are reduced such thatquantitation of hydrogen peroxide produced by the glucose oxidasereaction is not compromised. An enzyme composition may also includemultiple enzymes used for the detection of one (e.g., analyte glucose,enzyme composition glucose oxidase and mutarotase) or more analytes.Enzyme compositions for use in the practice of the present invention maybe from recombinant and/or synthetic sources. Typically, the enzyme ispresent in an amount of from about 10 units to about 5,000 units pergram of the total weight of the hydrogel.

An exemplary electrolyte is a salt, for example, a chloride salt,preferably, NaCl. Background signal in the hydrogels of the presentinvention can be determined by a number of standard methods. In thepresent invention, the background electrical signal is typically lessthan approximately 200 nA, preferably less than about 100 nA, morepreferably less than about 50 nA. Components of the hydrogel, may betreated to remove compounds that cause background electrical signal, forexample, using a diafiltration procedure to remove electroactivecompounds therefrom.

Hydrogel compositions of the present invention may include manufacturedsheets of hydrogel material as well as individual, essentially circularhydrogels.

In addition to the above components, the hydrogels may further comprisea buffering agent present in an amount sufficient to maintain a pH inthe hydrogel in a range of from about 3 to about 9, preferably in arange of about pH 6 to about pH 8, and more preferably the bufferingagent is sufficient to maintain a pH of about 7.4. An exemplary bufferis a phosphate buffer.

Hydrophilic compounds used to generate hydrogels are discussed hereinand include, but are not limited to, polyethylene oxide, polyacrylicacid, polyvinyl alcohol, polyacrylamidomethylpropane-sulfonate,copolymers thereof, and combinations thereof. As discussed herein, thehydrophilic compound may further comprise cross-linking agent(s), e.g.,bisacrylamide. The formulations of the present invention may be madewith or without a humectant. The hydrophilic compound may be present inan amount of less than about 40% by weight and water is present in anamount of more than 60% by weight based on the weight of the hydrogel,preferably, the hydrophilic compound which forms a gel is present in anamount in the range of from about 1% to about 25%, preferably in therange of about 5% to about 20%, more preferably about 10% to about 15%,based on total weight of the hydrogel. Alternatively, when a humectantis used, the hydrophilic compound is preferably in the range of fromabout 8% to about 12% based on total weight of the hydrogel containingthe humectant.

Further, the hydrogel may comprise a structural support materialembedded in the hydrogel. Examples of such support materials are givenherein. The support material may, for example, be a nonwoven material.Also as discussed herein, the hydrogel is typically substantially planarand has first and second surfaces, on which a mask layer, and/or gelretaining layer, and/or further release liners (e.g., see FIG. 1) may bedisposed. The hydrogel also has sufficient flexibility so as to conformto human skin.

The hydrogels are substantially planar and have a thickness in a rangeof about 1 mil to about 60 mils, preferably about 1 mil to about 25mils, more preferably about 5 mils to about 10 mils. In a preferredembodiment, the hydrogel has first and second surface areas, and eachsurface area is in a range of about 0.5 cm² to about 10 cm², morepreferably between about 0.5 cm² to about 2.5 cm², and the hydrogel hasa thickness of from about 1 mil to 10 mils. In a preferred embodiment, ahydrogel disk is about ¾ inch in diameter±15% (i.e., 0.44 sq. in.±0.07sq. in.) and has a thickness of about 5 mils.

In another aspect, the present invention relates to the discovery that acompound, e.g., a biocide, may be formulated into an ionicallyconductive material, even though the compound may interfere withdetecting an analyte in the ionically conductive material, because thepresence of the compound may be reduced by placing the ionicallyconductive material, comprising the compound, in contact with at leastone component comprised of a material that is partially permeable to thecompound, under conditions that allow the compound to migrate out of theionically conductive material and into the component—thus reducing thepresence of the compound in the ionically conductive material. In thisembodiment the ionically conductive material (ICM) comprising thecompound is placed in contact with the component or material (into whichit can migrate) under conditions and for a sufficient period of timeprior to use of the ionically conductive material in order to reduce theconcentration of the compound before use of the ICM. Following theguidance of the specification, in particular the Examples, suchconditions and times can be determined for any compound of interest(e.g., biocides). The ability of a selected compound to migrate into aselected material or component can be evaluated as described, forexample, in Examples 1, 2, and 3.

This discovery is useful, for example, in that biocide(s) (such as,undecylenates or parabens) can be used in the manufacturing stages of ahydrogel but can be removed from the hydrogel before its use indetecting the presence of a selected analyte. For example, where thecollection inserts are hydrogels (FIG. 1, 122, 124), the essentiallycircular hydrogel disks may be made from a water solution ofpolyethylene oxide, phosphate buffer, and glucose oxidase, impregnatedin a 0.004 inch thick nonwoven PET (e.g., Remay™ #2250 or Delnet™). Thiscomposite begins as roll stock from which circular discs are cut. Thesecircular disks (“hydrogels”) are then placed into contact with the maskand gel retaining layer materials as shown in FIG. 1 and subsequentlyused in collecting samples of analyte. During the manufacturing of thehydrogel disks concentrations of the biocide(s) effective to greatlyreduce or prevent growth of microorganisms can be used. Then, uponassembly of, for example, an autosensor where the hydrogels are now incontact with materials into which the biocides can migrate, the biocidescan migrate into such materials thus reducing the concentration of thebiocide in the hydrogel before use of the autosensor to detect analyteconcentration (by, for example, placing the autosensor into a monitoringsystem).

Accordingly, in one aspect of the present invention a method isdescribed for reducing a presence of a compound (e.g., a biocide) in anionically conductive material wherein, for example, the presence of thecompound interferes with detecting an analyte in the material. In oneembodiment, the method comprises placing the ionically conductivematerial (comprising the compound) in contact with at least one material(e.g., a component of a device capable of detecting the analyte) whereinthe material/component is at least partially permeable to the compound.Contact is maintained under conditions that allow the compound tomigrate out of the ionically conductive material and into thematerial/component, thus reducing the presence of the compound in theionically conductive material.

Exemplary biocides for use in the present invention include, but are notlimited to, undecylenic acid and phenolic compounds (e.g., parabens,such as an ester of p-hydroxybenzoic acid or mixtures thereof, suchesters may include methyl ester, ethyl ester, propyl ester, butyl ester,and isobutyl ester).

Exemplary materials into which such compounds may migrate include, butare not limited to, polyester(s), polyurethane(s), polyethylene(s),acrylic co-polymers, styrene butadiene copolymers, and mixtures thereof.

In one embodiment, the analyte of interest is glucose and the ionicallyconductive medium comprises part of a collection assembly capable ofbeing used in an iontophoretic sampling device, for example, thecollection assembly shown in FIG. 1. In this embodiment, the collectionassembly comprises, (i) a collection insert layer comprising theionically conductive material containing the compound, wherein theionically conductive material has a first surface and a second surface,(ii) a mask layer comprising a material that is substantiallyimpermeable to the selected analyte or derivatives thereof, wherein themask layer (a) has an inner face and an outer face and the inner face ispositioned in facing relationship with the first surface of thecollection insert, and (b) defines an opening that exposes at least aportion of the first surface of the collection insert layer, and (iii) aretaining layer having an inner face and an outer face wherein the innerface is positioned in facing relationship with the second surface of thecollection insert, and wherein the retaining layer defines an openingthat exposes at least a portion of the second surface of the collectioninsert layer. Such a mask layer and/or retaining layer can be comprisedof, for example, a polyurethane-like material or a polyester-likematerial, i.e., a material into which the compound can migrate.Exemplary materials into which such compounds may migrate include, butare not limited to, polyester(s), polyurethane(s), polyethylene(s),acrylic co-polymers, styrene butadiene copolymers, and mixtures thereof.Other liners (e.g., FIG. 1, 130, 132) used in such assemblies may bemade of materials permeable to the compound or of materials impermeableto the compound.

The present invention also includes methods of manufacturing hydrogelsand collections assemblies of the present invention. For example,producing hydrogels containing biocides and placing the hydrogels incontact with a material into which the biocides can migrate.

3. Exemplary Analytes

The analyte can be any specific substance or component that one isdesirous of detecting and/or measuring in a chemical, physical,enzymatic, or optical analysis. Such analytes include, but are notlimited to, amino acids, enzyme substrates or products indicating adisease state or condition, other markers of disease states orconditions, drugs of abuse (e.g., ethanol, cocaine), therapeutic and/orpharmacologic agents (e.g., theophylline, anti-HIV drugs, lithium,anti-epileptic drugs, cyclosporin, chemotherapeutics), electrolytes,physiological analytes of interest (e.g., urate/uric acid, carbonate,calcium, potassium, sodium, chloride, bicarbonate (CO₂), glucose, urea(blood urea nitrogen), lactate and/or lactic acid, hydroxybutyrate,cholesterol, triglycerides, creatine, creatinine, insulin, hematocrit,and hemoglobin), blood gases (carbon dioxide, oxygen, pH), lipids, heavymetals (e.g., lead, copper), and the like. In preferred embodiments, theanalyte is a physiological analyte of interest, for example glucose, ora chemical that has a physiological action, for example a drug orpharmacological agent.

In order to facilitate detection of the analyte, an enzyme can bedisposed within the one or more collection reservoirs. The selectedenzyme is capable of catalyzing a reaction with the extracted analyte tothe extent that a product of this reaction can be sensed, e.g., can bedetected electrochemically from the generation of a current whichcurrent is detectable and proportional to the amount of the analytewhich is reacted. In one embodiment of the present invention, a suitableenzyme is glucose oxidase, which oxidizes glucose to gluconic acid andhydrogen peroxide. The subsequent detection of hydrogen peroxide on anappropriate biosensor electrode generates two electrons per hydrogenperoxide molecule creating a current that can be detected and related tothe amount of glucose entering the device. Glucose oxidase (GOx) isreadily available commercially and has well known catalyticcharacteristics. However, other enzymes can also be used, as long asthey specifically catalyze a reaction with an analyte or substance ofinterest to generate a detectable product in proportion to the amount ofanalyte so reacted.

In like manner, a number of other analyte-specific enzyme systems can beused in the invention, which enzyme systems operate on much the samegeneral techniques. For example, a biosensor electrode that detectshydrogen peroxide can be used to detect ethanol using an alcohol oxidaseenzyme system, or similarly uric acid with urate oxidase system, ureawith a urease system, cholesterol with a cholesterol oxidase system, andtheophylline with a xanthine oxidase system.

In addition, the oxidase enzyme (used for hydrogen peroxidase-baseddetection) can be replaced with another redox system, for example, thedehydrogenase-enzyme NAD-NADH, which offers a separate route todetecting additional analytes. Dehydrogenase-based sensors can useworking electrodes made of gold or carbon (via mediated chemistry).Examples of analytes suitable for this type of monitoring include, butare not limited to, cholesterol, ethanol, hydroxybutyrate,phenylalanine, triglycerides, and urea. Further, the enzyme can beeliminated and detection can rely on direct electrochemical orpotentiometric detection of an analyte. Such analytes include, withoutlimitation, heavy metals (e.g., cobalt, iron, lead, nickel, zinc),oxygen, carbonate/carbon dioxide, chloride, fluoride, lithium, pH,potassium, sodium, and urea. Also, the sampling system described hereincan be used for therapeutic drug monitoring, for example, monitoringanti-epileptic drugs (e.g., phenytion), chemotherapy (e.g., adriamycin),hyperactivity (e.g., ritalin), and anti-organ-rejection (e.g.,cyclosporin).

Preferably, the biosensor electrode must be able to detect the analytewhich has been extracted into the one or more collection reservoirs whenpresent at nominal concentration levels. Suitable biosensor electrodesand associated sampling systems as described in are described in PCTPublication Nos. WO 97/10499, published 20 Mar. 1997 and WO 98/42252,published 1 Oct. 1998.

In one embodiment of the ionically conductive medium of the presentinvention, the hydrogel comprises approximately the followingproportions of components: 0.90 wt % sodium chloride, 0.22 wt % sodiumphosphate monobasic, 2.25 wt % sodium phosphate dibasic, 0.20 wt %sodium undecylenate, 10.0 wt % Polyox™-brand polyethylene oxide(approximately 600,000 MW, available from Union Carbide, Danbury Conn.),0.64 wt % glucose oxidase and 85.87 wt % purified water (note that thewt % of glucose oxidase can vary depending on the activity of theglucose oxidase typically 1,000 units of glucose oxidase is employed inthis formulation—adjustment in water wt % can be used to “round-out” thetotal wt % of the formulation). In another embodiment of the ionicallyconductive medium of the present invention, the hydrogel comprisesapproximately the following proportions of components: 0.90 wt % NaCl,0.32 wt % sodium phosphate monobasic, 2.07 wt % sodium phosphatedibasic, 0.20 wt % Nipastat® biocide, 10.0 wt % Polyox™-brandpolyethylene oxide (approximately 600,000 MW), 0.64 wt % glucose oxidaseand 85.87 wt % purified water (note that the wt % of glucose oxidase canvary depending on the activity of the glucose oxidase typically 1,000units of glucose oxidase is employed in this formulation—adjustment inwater wt % can be used to “round-out” the total wt % of theformulation). In yet another embodiment of the ionically conductivemedium of the present invention, the hydrogel comprises approximatelythe following proportions of components: 0.90 wt % NaCl, 0.26 wt %sodium phosphate monobasic, 2.17 wt % sodium phosphate dibasic—7H₂O,0.20 wt % Nipastat® biocide, 10.00 wt % Polyox™-brand polyethylene oxide(approximately 600,000 MW), 1.00 wt % bisacrylamide (2% solution),glucose oxidase to give 1000 units of enzymatic activity per gram of geland the remaining volume in purified water.

The concentration of the biocide is typcially based on the concentrationof the biocide wherein it acts effectively as a biocide. Thisconcentration can vary depending on the selected biocide and suitableconcentrations can be tested for efficacy as discussed herein. A typicalrange for the biocide concentration is about 0.01 wt % to 5 wt %,preferably between about 0.1 wt % to about 1 wt %, more preferablybetween about 0.2 wt % and 0.5 wt %.

4. Exemplary Sampling Systems

An automatic sampling system may be used to monitor levels of analyte,for example, glucose, in a biological system via the transdermallyextraction of the analyte (e.g., glucose) from the biological system,particularly an animal subject. Transdermal extraction is carried out byapplying an electrical current or ultrasonic radiation to a tissuesurface at a collection site. The electrical current is used to extractsmall amounts of glucose from the subject into a collection reservoir.The collection reservoir is in contact with a sensor element (biosensor)which provides for measurement of glucose concentration in the subject.As glucose is transdermally extracted into the collection reservoir, theanalyte reacts with the glucose oxidase within the reservoir to producehydrogen peroxide. The presence of hydrogen peroxide generates a currentat the biosensor electrode that is directly proportional to the amountof hydrogen peroxide in the reservoir. This current provides a signalwhich can be detected and interpreted (for example, employing analgorithm using statistical methods) by an associated system controllerto provide a glucose concentration value or amount for display.

In the use of the sampling system, a collection reservoir is contactedwith a tissue surface, for example, on the stratum corneum of asubject's skin. An electrical current is then applied to the tissuesurface in order to extract glucose from the tissue into the collectionreservoir. Extraction is carried out, for example, continually over aperiod of about 12 hours. The collection reservoir is analyzed, at leastperiodically, to measure glucose concentration therein. The measuredvalue correlates with the subject's blood glucose level.

To sample the analyte, one or more collection reservoirs are placed incontact with a tissue surface on a subject. The ionically conductivematerial within the collection reservoir is also in contact with anelectrode (for reverse iontophoretic extraction) which generates acurrent sufficient to extract glucose from the tissue into thecollection reservoir. Referring to FIG. 1, an exploded view of exemplarycomponents comprising one embodiment of an autosensor for use in aniontophoretic sampling system is presented. The autosensor componentsinclude two biosensor/iontophoretic electrode assemblies, 104 and 106,each of which have an annular iontophoretic electrode, respectivelyindicated at 108 and 110, which encircles a biosensor electrode 112 and114. The electrode assemblies 104 and 106 are printed onto a polymericsubstrate 116 which is maintained within a sensor tray 118. A collectionreservoir assembly 120 is arranged over the electrode assemblies,wherein the collection reservoir assembly comprises two hydrogel inserts122 and 124 retained by a gel retaining layer 126 and mask layer 128.Further release liners may be included in the assembly, for example, apatient liner 130, and a plow-fold liner 132. In an alternativeembodiment, the electrode assemblies can include bimodal electrodes. Apolyurethane mask layer 128 as described in PCT Publication No. WO97/10356, published 20 Mar. 1997, may be present. Other embodiments ofthe autosensor are described in WO 99/58190, “Collection Assemblies forTransdermal Sampling System,” T. E. Conn, et al.

The mask and retaining layers are preferably composed of materials thatare substantially impermeable to the analyte (e.g., glucose) to bedetected (see, for example, U.S. Pat. Nos. 5,735,273, and 5,827,183). By“substantially impermeable” is meant that the material reduces oreliminates analyte transport (e.g., by diffusion). The material canallow for a low level of analyte transport, with the proviso that theanalyte that passes through the material does not cause significant edgeeffects at the sensing electrode used in conjunction with the mask andretaining layers. Examples of materials that can be used to form thelayers include, but are not limited to polyester, polyester derivatives,other polyester-like materials, polyurethane, polyurethane derivativesand other polyurethane-like materials.

The components shown in exploded view in FIG. 1 are intended for use ina automatic sampling system which is configured to be worn like anordinary wristwatch, as described in PCT Publication No. WO 96/00110,published 4 Jan. 1996. The wristwatch housing can further includesuitable electronics (e.g., microprocessor, memory, display and othercircuit components) and power sources for operating the automaticsampling system. The sensing electrode can be a Pt-comprising electrodeconfigured to provide a geometric surface area of about 0.1 to 3 cm²,preferably about 0.5 to 2 cm², and more preferably about 1 cm². Thisparticular configuration is scaled in proportion to the collection areaof the collection reservoir used in the sampling system of the presentinvention, throughout which the extracted analyte and/or its reactionproducts will be present. The electrode composition is formulated usinganalytical- or electronic-grade reagents and solvents which ensure thatelectrochemical and/or other residual contaminants are avoided in thefinal composition, significantly reducing the background noise inherentin the resultant electrode. In particular, the reagents and solventsused in the formulation of the electrode are selected so as to besubstantially free of electrochemically active contaminants (e.g.,anti-oxidants), and the solvents in particular are selected for highvolatility in order to reduce washing and cure times.

The reactive surface of the sensing electrode can be comprised of anyelectrically conductive material such as, but not limited to,platinum-group metals (including, platinum, palladium, rhodium,ruthenium, osmium, and iridium), nickel, copper, silver, and carbon, aswell as, oxides, dioxides, combinations or alloys thereof. Somecatalytic materials, membranes, and fabrication technologies suitablefor the construction of amperometric biosensors were described byNewman, J. D., et al. (Analytical Chemistry 67(24), 4594–4599, 1995).

Any suitable iontophoretic electrode system can be employed, however itis preferred that a silver/silver chloride (Ag/AgCl) electrode system isused. The iontophoretic electrodes are formulated typically using twoperformance parameters: (1) the electrodes are capable of continualoperation for extended periods, preferably periods of up to 24 hours orlonger; and (2) the electrodes are formulated to have highelectrochemical purity in order to operate within the present systemwhich requires extremely low background noise levels. The electrodesmust also be capable of passing a large amount of charge over the lifeof the electrodes. With regard to continual operation for extendedperiods of time, Ag/AgCl electrodes are capable of repeatedly forming areversible couple which operates without unwanted electrochemical sidereactions (which could give rise to changes in pH, and liberation ofhydrogen and oxygen due to water hydrolysis). The Ag/AgCl electrode isthus formulated to withstand repeated cycles of current passage in therange of about 0.01 to 1.0 mA per cm² of electrode area. With regard tohigh electrochemical purity, the Ag/AgCl components are dispersed withina suitable polymer binder to provide an electrode composition which isnot susceptible to attack (e.g., plasticization) by components in thecollection reservoir, e.g., the hydrogel composition. The electrodecompositions are also formulated using analytical- or electronic-gradereagents and solvents, and the polymer binder composition is selected tobe free of electrochemically active contaminants which could diffuse tothe biosensor to produce a background current.

The automatic sampling system can transdermally extract the sample in acontinual manner over the course of a 1–24 hour period, or longer, usingreverse iontophoresis. The collection reservoir comprises an ionicallyconductive medium, preferably the hydrogel medium described hereinabove.A first iontophoresis electrode is contacted with the collectionreservoir (which is typically in contact with a target, subject tissuesurface), and a second iontophoresis electrode is contacted with eithera second collection reservoir in contact with the tissue surface, orsome other ionically conductive medium in contact with the tissue. Apower source provides an electric potential between the two electrodesto perform reverse iontophoresis in a manner known in the art. Asdiscussed above, the biosensor selected to detect the presence, andpossibly the level, of the target analyte (for example, glucose) withina reservoir is also in contact with the reservoir.

In practice, an electric potential (either direct current or a morecomplex waveform) is applied between the two iontophoresis electrodessuch that current flows from the first electrode through the firstconductive medium into the skin, and back out from the skin through thesecond conductive medium to the second electrode. This current flowextracts substances through the skin into the one or more collectionreservoirs through the process of reverse iontophoresis orelectroosmosis. The electric potential may be applied as described inPCT Publication No. WO 96/00110, published 4 Jan. 1996.

As an example, to extract glucose, the applied electrical currentdensity on the skin or tissue can be in the range of about 0.01 to about2 mA/cm². In order to facilitate the extraction of glucose, electricalenergy can be applied to the electrodes, and the polarity of theelectrodes can be, for example, alternated so that each electrode isalternately a cathode or an anode. The polarity switching can be manualor automatic.

When a bimodal electrode is used, during the reverse iontophoreticphase, the power source provides a current flow to the first bimodalelectrode to facilitate the extraction of the chemical signal into thereservoir. During the sensing phase, the power source is used to providevoltage to the first sensing electrode to drive the conversion ofchemical signal retained in reservoir to electrical signal at thecatalytic face of the sensing electrode. The power source also maintainsa fixed potential at the electrode where, for example hydrogen peroxideis converted to molecular oxygen, hydrogen ions, and electrons, which iscompared with the potential of the reference electrode during thesensing phase. While one sensing electrode is operating in the sensingmode it is electrically connected to the adjacent bimodal electrodewhich acts as a counter electrode at which electrons generated at thesensing electrode are consumed.

The electrode subassembly can be operated by electrically connecting thebimodal electrodes such that each electrode is capable of functioning asboth an iontophoretic electrode and counter electrode along withappropriate sensing electrode(s) and reference electrode(s), to createstandard potentiostat circuitry.

A potentiostat is an electrical circuit used in electrochemicalmeasurements in three electrode electrochemical cells. A potential isapplied between the reference electrode and the sensing electrode. Thecurrent generated at the sensing electrode flows through circuitry tothe counter electrode (i.e., no current flows through the referenceelectrode to alter its equilibrium potential). Two independentpotentiostat circuits can be used to operate the two biosensors. For thepurpose of the present invention, the electrical current measured at thesensing electrode subassembly is the current that is correlated with anamount of chemical signal corresponding to the analyte.

The detected current can be correlated with the subject's blood glucoseconcentration (typically using statistical algorithms associated with amicroprocessor) so that the system controller may display the subject'sactual blood glucose concentration as measured by the sampling system.For example, the system can be calibrated to the subject's actual bloodglucose concentration by sampling the subject's blood during a standardglucose tolerance test, and analyzing the blood glucose using both astandard blood glucose monitor and the sampling system of the presentinvention. In addition or alternately, the sampling system can becalibrated at a calibration time point where the signal obtained fromthe sampling system at that time point is correlated to blood glucoseconcentration at that time point as determined by direct blood testing(for example, glucose concentration can be determined using a HemoCue®clinical analyzer (HemoCue AB, Sweden)). In this manner, measurementsobtained by the sampling system can be correlated to actual values usingknown statistical techniques. Such statistical techniques can beformulated as algorithm(s) and incorporated in a microprocessorassociated with the sampling system.

Further, the sampling system can be pre-programmed to begin execution ofits signal measurements (or other functions) at a designated time. Oneapplication of this feature is to have the sampling system in contactwith a subject and to program the sampling system to begin sequenceexecution during the night so that it is available for calibrationimmediately upon waking. One advantage of this feature is that itremoves any need to wait for the sampling system to warm-up beforecalibrating it.

5. Selectively Permeable Barriers

Further aspects of the present invention include, methods of generatinga selectively permeable barrier on an electrode surface, as well as,further means for reducing the presence of a compound in an ionicallyconductive material. In one embodiment, the presence of the compoundinterferes with detecting an analyte in the material. Previously it hasbeen required that the membrane film (i.e., selectively permeablebarrier) be formed a priori on an electrode and this represents anadditional step during the fabrication of a biosensor assembly. Thisrepresents a key disadvantage of the technique as it has been practicedheretofore. Experiments performed in support of the present inventiondemonstrate a one-step method for the formation of a permselectivemembrane to reduced interferences. For example, in the context ofglucose detection, glucose entering the hydrogel is converted to H₂O₂,which diffuses through the membrane film with little or no attenuation,whereas larger interfering molecules, such as uric acid andacetaminophen, are significantly attenuated, resulting in an enhancedselectivity of the H₂O₂ (from enzymatic oxidation of glucose) responseat the sensor surface.

Accordingly, in one aspect of the invention, interfering species arereduced by polymerizing an interfering compound to form anelectrochemically-inactive but permeation selective barrier at thereactive face of the sensor means. The permeation selectivecharacteristics of the polymer barrier can provide the added benefit ofreducing signals generated from interferants other than the speciesbeing polymerized. Because the aforementioned permeation selectivebarrier is created on the reactive face of the sensor means in situ,rather than prior to construction of the collection assembly, thepresent invention provides efficient means for manufacturing collectionassemblies that use this method for reducing the presence of aninterferant compound.

Examples 4, 5 and 6 describe the polymerization of compounds, e.g.,biocides, and formation of a polymer barrier (polymer film) at thereactive face of a sensor electrode. The polymer barrier formed has beenshown to selectively screen some interfering species (molecules), whileat the same time allowing accurate quantitation of an analyte ofinterest. In addition to generating a selectively permeable barrier,polymerization of the compound also serves to reduce the concentrationof the compound in the ionically conductive media.

In one aspect of the present invention, the the ionically conductivematerial, comprising the compound, is placed in contact with a reactiveface of a sensor element such that, when an electric current is flowingto the sensor element, the current flows through the ionicallyconductive material. The sensor element is then activated to provide theelectrical current for a period of time and under conditions sufficientto polymerize the compound on the reactive face of the sensor element,thus reducing the presence of the compound in the ionically conductivematerial. Such times and conditions can be determined for a variety ofcompounds, for example, methyl parabens, following the guidance of thespecification and in particular the methods illustrated in Examples 4, 5and 6.

The present invention also provides a method of forming a permeationselective barrier in situ on a reactive face of a sensor element. Inthis aspect of the invention, an ionically conductive material isformulated comprising a compound, for example, a phenolic compound,capable of polymerizing under the influence of an electrical current.The ionically conductive material is placed in contact with the reactiveface of a sensor element such that when the electric current is flowingto the sensor element, the current flows through the ionicallyconductive material. The sensor element is activated to provide theelectrical current for a period of time and under conditions sufficientto polymerize the compound on the reactive face of the sensor. Suchpolymerization serves to form a permeation selective barrier. In thecase of a biocide, the polymerization also serves to reduce theconcentration of the biocide in the ionically conductive material.

In a preferred embodiment of the present invention, the compound is abiocide, for example, a phenolic compound. Such a phenolic compound may,for example, be an ester of p-hydroxybenzoic acid, or mixture of suchesters (e.g., methyl ester, ethyl ester, propyl ester, butyl ester, andisobutyl ester). Related biocides are discussed herein above.

Sensor elements useful in the practice of the present invention havealso been described above. In a preferred embodiment the sensor elementis a platinum/carbon electrode.

Numerous analytes are discussed herein, an exemplary analyte beingglucose (see Examples 4, 5, and 6).

The present invention also includes collection assemblies for use insampling systems. Typically, a collection insert layer comprises anionically conductive material having a compound that will polymerize ona reactive face of a sensor element. The collection insert is placed inworking, i.e., functional, relationship with the reactive face. Such acollection insert may be part of an autosensor assembly and may includea support tray as well (see, e.g., FIG. 1).

Also included in the present invention are methods of manufacturing suchcollection assemblies (or autosensor assemblies). Such methods includeformulating the ionically conductive medium to contain the compound,wherein the ionically conductive material has a first surface and asecond surface. The first surface of the ionically conductive medium isthen placed in contact with a mask layer. Mask layers were discussedabove and typically comprise a material that is substantiallyimpermeable to the selected analyte or derivatives thereof. The masklayer (i) has an inner face and an outer face and the inner face ispositioned in facing relationship with the first surface of theionically conductive medium, and (ii) defines an opening that exposes atleast a portion of the first surface of the ionically conductive medium.The second surface of the ionically conductive medium is contacted witha retaining layer. The retaining layer has an inner face and an outerface wherein the inner face is positioned in facing relationship withthe second surface of the ionically conductive medium. The retaininglayer defines an opening that exposes at least a portion of the secondsurface of the ionically conductive medium to form the collectionassembly. The ionically conductive media may further comprise an enzymecomposition and other components as discussed above. For example, theionically conductive media may be hydrogels comprising an enzyme capableof reacting with an analyte to produce hydrogen peroxide, and a phenoliccompound that will polymerize under an electric current. The method mayfurther include placing a sensor element in operative contact with theionically conductive media (e.g., collection insert layer). In oneembodiment, upon application of electrical energy, the sensor elementreacts electrochemically with the phenolic compound to provide aselectively permeable barrier at an interface between the sensor elementand the collection insert layer. Other components (such as a supporttray) may be added during the manufacturing method, such as, thecomponents shown in FIG. 1 and discussed above.

The present invention also includes devices (e.g., collectionassemblies, laminates, and/or autosensors) made by these methods.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that thedescription above as well as the examples which follow are intended toillustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All patents, patent applications, and publications mentioned herein,both supra and infra, are hereby incorporated by reference.

EXPERIMENTAL

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperature, etc.) but someexperimental error and deviation should be accounted for. Unlessindicated otherwise, temperature is in degrees C. and pressure is at ornear atmospheric.

Example 1 Stability of the Nipastat® Biocide in the Hydrogel

Nipastat®-containing hydrogels were formulated with the appropriatebuffer salts under standard conditions, from which samples were takenfor analysis. After exposure to controlled environmental conditions(temperature and humidity) for differing periods of time, the samplehydrogels were extracted in acetonitrile (ACN)/water and assayed for thepresence of methyl, ethyl, propyl and butyl esters of p-hydroxybenzoicacid using a reverse phase octadecylsilane (ODS) HPLC column as follows.The hydrogels were cut using a ¾ inch punch to generate the sample disksto assay. Each disk was added to 5 mL of acetonitrile/water (30% ACN,v/v) and the Nipastat® biocide was extracted for 1 hour while shaking onan orbital shaker at 100 rpm. The extract was then filtered through a0.2 μm membrane prior to HPLC separation and UV detection at 254 nm.

Reverse phase HPLC chromatography was performed using a Waters 3.9 mm×15cm Nova-Pak C-18 column (Milford, Mass.) operating at a flow rate ofbetween about 1.0 to 2.0 mL/min. at 35° C. The eluent was monitored byUV at 254 nm using a Shimadzu SPD-10AU UV-Visible spectrometer (Kyoto,Japan). Samples 10 μL in volume were injected into the columnequilibrated in 30% ACN in water, and the following 20-minute gradientprogram was performed:

-   1. Initial time of injection: 30% ACN/70% water at a flow rate of 1    mL/min-   2. Linear Gradient from 30% ACN to 80% ACN for 9.0 minutes (1    mL/min)-   3. Linear Gradient from 80% ACN to 100% ACN for 0.5 minutes (1    mL/min)-   4. 100% ACN for 4.5 minutes (1 mL/min)-   5. Linear Gradient from 100% ACN to 30% ACN for 0.5 minutes at a    flow rate of 2 mL/min-   6.30% ACN for 5.5 minutes at a flow rate of 1 mL/min

The HPLC profiles of the extracts were compared with that of a standardsolution of known concentration comprising the p-hydroxybenzoatederivatives. The peak intensities of the experimental samples arecompared to those of the standards to determine relative amounts of eachextracted paraben. Typical retention times for the different esters ofp-hydroxybenzoic acid were determined to be: 2.4 to 2.9 minutes forp-hydroxybenzoic acid methyl ester (methylparaben), 4.0 to 4.8 minutesfor p-hydroxybenzoic acid ethyl ester (ethylparaben), 6.0 to 7.0 minutesfor p-hydroxybenzoic acid propyl ester (propylparaben), and 7.5 to 8.5minutes for the p-hydroxybenzoic acid butyl esters (butylparaben andisobutylparaben). The data (Table 1, presented) indicate that theNipastat® biocide is stable within the hydrogel, so any short term lossof compound is not due to degradation.

TABLE 1 Stability of Nipastat ® Biocide within Gel Laminate BiocidePercent of Time Relative Content Theoretical Point Temperature Humidity(%) Concentration (%) 0 40° C. 75% 0.15 75 2 weeks 40° C. 75% 0.14 70 1month 40° C. 75% 0.13 65 3 months 40° C. 75% 0.11 55 6 months 40° C. 75%0.11 55 0 25° C. 60% 0.15 75 1 month 25° C. 60% 0.14 70 3 months 25° C.60% 0.13 65 6 months 25° C. 60% 0.11 57

Example 2 Migration of the Nipastat® Biocide into Components of theBiosensor Collection Assembly

Collection assemblies incorporating Nipastat® biocide-comprisinghydrogels were assayed for retention of the biocide within the hydrogel.Samples of the hydrogel prior to and after incorporation into thecollection assembly, as well as the components of the collectionassembly in contact with the hydrogel, were prepared as described inExample 1. The decreasing quantities of parabens extracted from thehydrogel indicate that, over time, the Nipastat® biocide migrates out ofthe hydrogel and into the adjacent collection assembly components. Whenthe components of the collection assembly in contact with the hydrogel(liners, gel retaining layer, and mask) were assayed, it was determinedthat the Nipastat® biocide was migrating preferentially into the gelretaining layer (comprising a polyester derivative) and the mask layer(comprising a polyurethane derivative). These results are presented inTables 2 and 3, respectively. The liners (one comprising a polyethylenederivative and the other comprising a polypropylene derivative) showednegligible adsorption of the Nipastat® paraben components.

TABLE 2 Adsorption of Nipastat ® Paraben Components by Mask LayerComponent of Collection Assembly after 48 Hours at 4° C. Methyl EthylPropyl Butyl Gel Control 1 282756 93771 27621 75858 2 265561 58684 2593668194 3 303549 69162 28226 79360 4 199735 43754 18744 50220 5 23349552495 22146 59177 Average 257019 63573 24535 66562 Std. Dev. 41034 192524011 11984 Gel + Mask 1 5445 0 0 0 2 11776 285 0 0 3 13807 0 252 0 411268 0 0 436 5 8081 0 0 0 Average 10075 57 50 87 Std. Dev. 3304 127 113195 % Loss of Nipastat ® 96.1 99.9 99.8 99.9 biocide from Gel

TABLE 3 Adsorption of Nipastat ® Paraben Components by Gel RetainingLayer (GRL) Component of Collection Assembly after 9 Days at 4° C.Methyl Ethyl Propyl Butyl Gel Control 1 313086 70588 30407 82288 2256304 56529 24349 67760 3 250006 56747 22180 61920 4 261836 58534 2403466552 5 315315 68190 30121 82337 Average 279309 62118 26218 72171 Std.Dev. 32135 6737 3786 9511 Gel + GRL 1 147682 23429 4958 1377 2 23935643116 11465 15432 3 231307 38410 9664 8640 4 251620 46214 12662 17787 5289836 53073 15618 20353 Average 231960 40848 10873 12718 Std. Dev.52187 11099 3953 7691 % Loss of Nipastat ® 17.0 34.2 58.5 82.4 biocidefrom Gel

Example 3 Migration of the Biocide Sodium Undecylenate into Componentsof the Biosensor Collection Assembly

Collection assemblies incorporating sodium undecylenate-comprisinghydrogels were assayed for retention of the biocide within the hydrogel.The presence of undecylenic acid in the hydrogel and assembly componentswas determined by gas chromatography (GC) using a Hewlett Packard(Avondale, Pa.) 5890 gas chromatogram equipped with an HP 3396Aintegrator. The hydrogels and collection assembly components were cutusing a ¾ inch punch to generate the samples to assay. Each sample“disk” was added to 4 mL of 1M HCl, and the undecylenic acid wasextracted for 2 hours while shaking on an orbital shaker at 150 rpm,followed by 10 minutes at 100 rpm. The sample disks were then extractedtwice with 4 mL of ethyl acetate. Samples 1 μL in volume were injectedinto the GC and the results compared to that generated for a standardsolution of undecylenic acid. The undecylenic acid was also shown tomigrate out of the hydrogel and into the adjacent collection assemblycomponents over time, as indicated by decreasing quantities of biocideextracted from the hydrogel two weeks after incorporation into thecollection assembly (Table 2.) Hydrogels comprising undecylenic acidexposed solely to the polyurethane mask component, or the polyester gelretaining layer also demonstrated loss of undecylenic acid from thehydrogel over time.

TABLE 4 Adsorption of Undecylenic Acid into Mask Layer and Gel RetainingLayer (GRL) Components of Collection Assembly after Two Weeks at RoomTemperature Hydrogel % Loss for Gel % Loss for Gel + % Loss for Gel +Sample Control Mask Layer GRL 1 37.2 78.2 22.2 2 52.8 72.5 24.7 3 64.973.3 37.0 4 68.1 68.5 28.4 5 75.2 6 76.2 Average 62.4 73.1 28.1 Std.Dev. 15.0 4.0 6.5

Example 4 Electropolymerization of 0.2% Nipastat® Biocide Directly ontoPt/C Electrodes

The polymerization of the Nipastat® biocide and formation of a polymerbarrier (polymer film) at the reactive face of the Pt/C sensor electrodewas demonstrated as follows. Experiments were performed using a BAS100W/B potentiostat (Bioanalytical Systems, West Lafayette Ind.). Theelectropolymerization reactions were initiated by either (a) cycling theelectrode immersed in 0.2% Nipastat® biocide solution between −0.2 and1.0V vs. Ag/AgCl, or (b) by applying a constant potential (0.77V vs.AgCl) for 10 to 40 minute intervals. The modified electrodes wereimmersed in phosphate buffer (pH 7.4) overnight to remove loosely boundmaterial. The samples were removed from the buffer solutions, rinsedgently with distilled water, and allowed to dry prior to use.

The following functional test was used to determine the sensitivity ofthe sensors element exposed to a Nipastat® biocide-containing solution,as compared to control sensing elements upon addition of glucose. Thesensing element with the Nipastat®-derived polymer film was combinedwith the collection assembly comprising the ionically conductivematerial to form the autosensor assembly. The autosensor assembly wasthen preconditioned for 10 minutes at 0.77V, followed by 50 minutes at0.42V. A glucose solution (200 μM) was deposited onto the ionicallyconductive material; this is preferably achieved by placing a circularabsorbant disk, or “wick,” against the ionically conductive medium tospread the glucose solution evenly across the surface of the material.The response of the sensor element is measured from this time forward.

Irreversible deposition of the Nipastat® biocide onto the sensingelement was confirmed independently by comparing the response of thesensing element to 1 mM ferricyanide before and afterelectropolymerization of the biocide. The potential required forpolymerization of the Nipastat® biocide onto the reactive face of thesensing element was determined to be between about 0.25V and about 1.0V,preferably between about 0.6V and about 0.9 V, most preferably at about0.9V. Approximately 90% of the reactive face of the sensor element wasblocked by polymerized biocide upon exposure of the sensor element to0.77V for 10 minutes.

Example 5 In Situ Polymerization of the Nipastat® Biocide onto theSensing Electrode

The effectiveness of in situ formation of a phenolic compound-derivedelectropolymerized barrier (polymer film) at the reactive face of thePt/C sensor electrode was demonstrated by measuring the response of theunderlying Pt/C sensor electrode after deposition of knownconcentrations of model compounds on the hydrogels prepared in thepresence and absence of the Nipastat® paraben compounds (FIG. 2). Thelist of model compounds tested included glucose (200 μM), hydrogenperoxide (200 μM), uric acid (100 μM), and acetaminophen (230 and 331μM). The response of the sensor elements was measured upon exposure ofthe electrode to 200 μM glucose in the presence or absence of the modelcompound, in a manner similar to that used in Example 4. The sensingelements were combined with the collection assembly comprising theionically conductive material containing the biocide, to form theautosensor assembly. The autosensor assembly was then preconditioned for10 minutes at 0.77V, followed by 50 minutes at 0.42V, during which thepolymerization of the biocide occurs. The glucose solution (200 μM) plusthe compounds to be analyzed were deposited onto the ionicallyconductive material, and the response of the sensor element measuredfrom this time forward.

Table 5 demonstrates the time-dependent responses of the collectionassemblies to the test compounds. The in situ membrane film formed bypolymerization of the Nipastat® biocide at the reactive face of thesensor electrode attenuated the response of the collection assembly tothe uric acid and acetaminophen. However, in situ formation of themembrane film had little impact on the response generated by addition ofhydrogen peroxide or glucose. Thus, the in situ membrane filmdemonstrated selectivity with respect to the permeation properties (a“permselective” barrier).

TABLE 5 Responses of Pt/C-Sensing Electrode to Model Compounds Charge(nC) Hydrogel After After After Compound Composition 2.5 min 5.0 min 7.0min H₂O₂ Standard 81766 ± 10019 160916 ± 17594  209663 ± 20396  H₂O₂Nipastat ®-containing   63398 ± −7849  126747 ± 13723  168126 ± 16407 Glucose Standard 50814 ± 3768  107464 ± 6035  146066 ± 7149  GlucoseNipastat ®-containing 37598 ± 2190  82402 ± 4006  114781 ± 4902  Uricacid Standard 72630 ± 5491  135108 ± 6605  162773 ± 5640  Uric acidNipastat ®-containing 19558 ± 4007  47146 ± 9357  66494 ± 12520Acetaminophen Standard 177161 ± 29100  366212 ± 45477  464431 ± 45109 Acetaminophen Nipastat ®-containing 40752 ± 15503 102906 ± 38278  150029± 54509 

Table 6 illustrates a similar observation at three discrete time points(2.5, 5.0 and 7.0 minutes, respectively) after application of thepolarizing potential (i.e. after 60 minutes of “preconditioning”). Theselectivity of the collection assembly into which hydrogels comprisingNipastat® biocide were incorporated indicates that the glucose andhydrogen peroxide responses are being retained while the uric acid andacetaminophen responses are reduced.

TABLE 6 Selectivity for Glucose against Interferants using Pt/C-sensingElectrode-Hydrogel System Selectivity Ratio Hydrogel After After AfterCompound Composition 2.5 min 5.0 min 7.0 min Uric acid Standard 1.431.26 1.11 Uric acid Nipastat ®-containing 0.52 0.57 0.58 AcetaminophenStandard 3.49 3.41 3.18 Acetaminophen Nipastat ®-containing 1.08 1.251.31

The selectivity of the collection assembly can be expressed by the useof a selectivity ratio. The ratio is defines as the response (i.e.charge) generated by the interfering species divided by the response(charge) generated by the analyte, which in this embodiment is glucose.

${{Selectivity}\mspace{14mu}{Ratio}} = \frac{{{Response}({charge})}\mspace{11mu}{of}\mspace{14mu}{interferant}}{{Response}\;({charge})\mspace{11mu}{of}\mspace{14mu}{{analyte}({glucose})}}$

The smaller the selectivity ratio, the more selective a collectionassembly is for the analyte. This ratio will be decreased (indicating amore selective analyte measurement) for high sensor responses to analyte(glucose) or for low responses to the interferant. Selectivity ratiosfor the interferants uric acid and acetaminophen are shown in Table 6.Comparison of the selectivity ratios shows improvement in theselectivity when Nipastat® biocide-comprising hydrogels are used in thecollection assemblies for the detection of glucose. These datademonstrate that, in fluids containing electroactive interferants, thein situ formation of a membrane film provides an effective method forselective measurement of analytes such as, but not limited to, glucose.Additional factors which were evaluated for their effect of the efficacyof interferant response suppression included the sensitivity of thesensor electrode, the presence of surfactants and the duration of thepreconditioning time at 0.77V. Interferant signal response was moreattenuated with increasing sensitivity of the sensor electrode. Additionof a surfactant to either the hydrogel composition or to the reactivesurface of the sensor element also led to attenuated interferant signalresponse. The attenuation in signal was determined to not be due todegradation of the glucose oxidase enzyme for the hydrogels comprisingboth surfactant and Nipastat® biocide.

The collection assemblies were preconditioned for different lengths oftime before glucose or acetaminophen were deposited on the hydrogel. Theresponse in the presence of acetaminophen decreased significantly withthe length of the preconditioning time while the values for glucoseremained constant. These results indicate that preconditioning timesfrom zero minutes to about 1 hour, and more particularly from about 5minutes to about 30 minutes led to in situ polymerization of theNipastat® biocide and suppression of interferant signal without any lossin glucose response. The result is consistent with polymerization anddeposition of the Nipastat®-based film as a function of time.

Example 6 Response of the Sensing Electrode to Glucose, Acetominophenand Uric Acid in the Presence of Nipastat® Biocide Versus UndecylenicAcid

Two biocides were compared with respect to the effects their presence(within the ionically conductive medium) had on signal generation at thesensing element under three conditions: in the presence of glucose,glucose plus acetaminophen, and glucose plus uric acid. The Nipastat®biocide was shown in the examples above to form a permeation selectivebarrier at the reactive face of the sensor element. The second biocidetested, undecylenic acid, was not expected to polymerize under theiontophoretic conditions used during the functionality test as describedin the previous example. The measured responses of the sensor elementsare presented in Table 7, normalized to a control “background” responsefor each sensor element. As described in Example 5, the sensorelectrodes were assembled with the collection insert layer containingthe appropriate ionically conductive material (control, Nipastat®biocide, or undecylenic acid biocide) to form the autosensor assemblies.The autosensor assemblies were preconditioned for 10 minutes at 0.77V,followed by 50 minutes at 0.42V, after which the glucose solution (200μM) plus or minus the interferant species was deposited onto theionically conductive material, and the response of the sensor elementmeasured from this time forward. The results confirm that Nipastat®biocide forms a permeation selective barrier which selectively impedesacetominophen and uric acid signal generation, while the undecylenicacid does not. The higher background measurements seen in the presenceof Nipastat® biocide and undecylenic acid, indicating that thesecompounds are electrochemically active, and thus could potentially actas interferants themselves.

TABLE 7 Response of the Sensing Electrode to Glucose, Acetominophen andUric Acid in the Presence of Nipastat ® Biocide versus SodiumUndecylenate Percent Recovery in the Presence of 200 μM Glucose Gel +Gel + Control Gel Nipastat ® Na-Undecylate Sample Replicate (%Recovery)* (% Recovery)* (% Recovery)* Glucose alone 1 40.8 25.0 38.9Glucose alone 2 39.3 28.9 49.2 Glucose alone 3 31.3 46.8 48.7 Glucosealone 4 40.2 36.1 52.7 Glucose alone 5 38.2 27.3 53.9 Glucose alone 639.9 28.4 53.1 Average 38.3 32.1 49.4 Standard Deviation 3.5 8.1 5.6Background (nA) 33.8 59.5 79.8 331 uM acetaminophen 1 137.9 77.7 143.8331 uM acetaminophen 2 138.6 98.2 155.6 331 uM acetaminophen 3 136.446.3 106.9 331 uM acetaminophen 4 145.8 78.2 86.5 331 uM acetaminophen 5123.3 53.9 114.3 331 uM acetaminophen 6 127.4 60.9 153.0 Average 134.969.2 126.7 Standard Deviation 8.2 19.1 28.2 Background (nA) 27.7 55.775.8 100 uM uric acid 1 77.4 48.8 72.4 100 uM uric acid 2 73.2 43.5 62.6100 uM uric acid 3 68.3 60.7 75.7 100 uM uric acid 4 71.9 31.8 80.0 100uM uric acid 5 56.8 55.9 — 100 uM uric acid 6 72.5 54.7 — Average 70.049.3 72.7 Standard Deviation 7.1 10.4 7.4 Background (nA) 32.3 62.5111.8 *Percent Recoveries (% recovery) were computed at five minutesafter deposition of sample.

Example 7 Microbial Challenge of Ionically Conductive Media in thePresence of Nipastat® Biocide Versus Undecylenic Acid

Nipastat® biocide and undecylenic acid were compared with respect to theeffects their presence (within the ionically conductive medium) had onmicrobial growth over time. A modified USP antimicrobial preservativeeffectiveness test was performed, using Aspergillus niger, Candidaalbicans, Eschericia coli, Pseudomonas aeruginosa and Staphylococcusaureus. Separate portions of the two hydrogels were inoculated with alow concentration of one of the listed microorganisms, and recovery ofthe microorganism was determined over a period of 28 days. For thisassay, the microorganisms were cultured, harvested and diluted to yieldworking suspensions of 2.0×10³ to 2.0×10⁴ colony forming units(CFUs)/sample. Both the Nipastat® biocide and the undecylenic acidretained their biocide activity within the hydrogel, and were shown tobe effective at reducing the microbial count across the 28 day periodtested. The undecylenic acid was more effective than the Nipastat®biocide against the Aspergillus niger, Pseudomonas aeruginosa andStaphylococcus aureus inoculations. Both biocides were equally effectiveversus the Candida albicans and Eschericia coli inoculations.

1. A method of manufacturing a collection assembly for use in a glucosesampling system and for electrochemical detection of an amount orconcentration of glucose, the method comprising: providing a hydrogelcomprising: polyethylene oxide present in an amount of about 5% to about20% by weight based on the total weight of the hydrogel; water in anamount of about 50% or more and about 95% or less based on the totalweight of the hydrogel; a chloride salt, wherein a background electricalsignal in the gel is less than approximately 200 nA; a phosphate bufferin an amount sufficient to maintain a pH in the hydrogel in the range of6 to 8; an enzyme composition comprising glucose oxidase, said glucoseoxidase present in an amount of from about 10 units to about 5,000 unitsper gram of the total weight of the hydrogel; and a biocide comprisingan undecylenate, wherein said hydrogel comprises a first surface and asecond surface; contacting the first surface of the hydrogel with a masklayer, the mask layer comprising a material that is substantiallyimpermeable to glucose or derivatives thereof, wherein the mask layer(i) has an inner face and an outer face, and the inner face ispositioned in facing relationship with the first surface of thehydrogel, and (ii) defines a first opening that exposes at least aportion of the first surface of the hydrogel, and contacting the secondsurface of the hydrogel with a gel retaining layer, the gel retaininglayer comprising an inner face and an outer face, wherein the inner faceis positioned in facing relationship with the second surface of thehydrogel, and wherein the gel retaining layer defines a first openingthat exposes at least a portion of the second surface of the hydrogel tocomplete the collection assembly.
 2. The method of manufacturing ofclaim 1, wherein said collection assembly is formed as a laminate. 3.The method of manufacturing of claim 1, further comprising positioning arelease liner in contact with the mask layer.
 4. The method ofmanufacturing of claim 3, further comprising positioning a plow-foldliner in contact with the gel retaining layer.
 5. The method ofmanufacturing of claim 1, wherein the polyethylene oxide of saidhydrogel comprises crosslinking.
 6. The method of manufacturing of claim5, wherein said crosslinking is achieved by thermal reaction, chemicalreaction, or providing ionizing radiation.
 7. The method ofmanufacturing of claim 6, wherein said ionizing radiation is provided byelectron beam radiation, UV radiation, or gamma radiation.
 8. The methodof manufacturing of claim 1, wherein said biocide comprises undecylenicacid, a salt of undecylenic acid, or mixtures thereof.
 9. The method ofmanufacturing of claim 1, wherein said background electrical signal isless than approximately 50 nA.
 10. The method of manufacturing of claim1, wherein said enzyme composition further comprises a mutarotaseenzyme.
 11. The method of manufacturing of claim 1, wherein (i) saidglucose oxidase which catalyzes a reaction between glucose and oxygenresulting in the generation of hydrogen peroxide, and (ii) hydrogenperoxide degradative components of the hydrogel are reduced.
 12. Themethod of manufacturing of claim 1, wherein said hydrogel furthercomprises a structural support material embedded in the hydrogel. 13.The method of manufacturing of claim 1, wherein one or more componentsof said hydrogel are treated to remove compounds that cause backgroundelectrical signals.
 14. The method of manufacturing of claim 13, whereinone or more of said hydrogel components are treated using adiafiltration procedure to remove electroactive compounds therefrom. 15.The method of manufacturing of claim 1, wherein said hydrogel furthercomprises bisacrylamide.
 16. The method of manufacturing of claim 1,wherein said biocide is present in the hydrogel at a concentration ofbetween about 0.01% to about 5% based on the total weight of thehydrogel.
 17. The method of manufacturing of claim 16, wherein saidbiocide is present in the hydrogel at a concentration of between about0.1% to about 1% based on the total weight of the hydrogel.
 18. Themethod of manufacturing of claim 17, wherein said biocide is present inthe hydrogel at a concentration of between about 0.2% to about 0.5%based on the total weight of the hydrogel.
 19. The method ofmanufacturing of claim 1, wherein the collection assembly furthercomprises a second hydrogel having a first surface and a second surface,said second hydrogel comprising: polyethylene oxide present in an amountof about 5% to about 20% by weight based on the total weight of thehydrogel; water in an amount of about 50% or more and about 95% or lessbased on the total weight of the hydrogel; a chloride salt, wherein abackground electrical signal in the gel is less than approximately 200nA; a phosphate buffer in an amount sufficient to maintain a pH in thehydrogel in the range of 6 to 8; an enzyme composition comprisingglucose oxidase, said glucose oxidase present in an amount of from about10 units to about 5,000 units per gram of the total weight of thehydrogel; and a biocide comprising an undecylenate; said inner face ofthe mask layer (i) is positioned in facing relationship with the firstsurface of the second hydrogel, and (ii) defines a second opening thatexposes at least a portion of the first surface of the second hydrogel;and said inner face of the gel retaining layer (i) is positioned infacing relationship with the second surface of the second hydrogel, and(ii) defines a second opening that exposes at least a portion of thesecond surface of the second hydrogel.